嵌合犬瘟热病毒H基因的重组狂犬病病毒的构建及免疫原性研究

doi:10.16431/j.cnki.1671-7236.2022.12.026

摘要/Abstract

摘要： 【目的】构建表达犬瘟热病毒（CDV）血凝素蛋白H基因的重组狂犬病病毒（RABV），并研究其生物学特性及免疫原性。【方法】在RABV HEP-dG株基因组G与L基因之间插入CDV H基因，构建重组全长cDNA质粒pHEP-dG （H）。将pHEP-dG （H）与辅助质粒共同转染BHK细胞，拯救携带CDV H基因的重组RABV HEP-dG （H）。将重组病毒接种BHK细胞，分析病毒的扩散能力；将重组病毒接种小鼠，分析病毒的致病性和免疫原性。【结果】RT-PCR及直接免疫荧光显示，传至第3代的病毒仍能检测到H基因，表明CDV H基因已成功插入RABV基因组中，并在HEP-dG （H）中稳定遗传和正确表达。HEP-dG （H）在BHK细胞中的生长曲线与HEP-dG毒株相似，病毒滴度在96 h达到峰值，但HEP-dG （H）的滴度在每个时间点略低于HEP-dG。以感染复数（MOI）为0.005感染BHK细胞，HEP-dG （H）的扩散能力比HEP-dG毒株低。HEP-dG （H）与HEP-dG对6周龄成年小鼠均不致死，但HEP-dG （H）对成年小鼠体重的影响弱于HEP-dG。HEP-dG （H）与HEP-dG均能诱导小鼠产生抗RABV的中和抗体，免疫后7 d抗体已达到保护水平（0.5 EU/mL）；此外，HEP-dG （H）可诱导产生CDV中和抗体。HEP-dG （H）与HEP-dG免疫小鼠3周后，均能抵御RABV标准攻毒毒株CVS-24的攻击。【结论】本研究成功构建重组RABV HEP-dG （H），其具有良好的免疫原性和安全性，可作为RABV-CDV新型二联基因工程候选疫苗。

关键词: 狂犬病病毒(RABV); 犬瘟热病毒(CDV); H基因; 狂犬病灭活疫苗(dG株); 免疫原性

Abstract: 【Objective】 The aim of this experiment was to construct recombinant Rabies virus (RABV) expressing the hemagglutinin H gene of Canine distemper virus (CDV), and study its biological characteristics and immunogenicity.【Method】 H gene of CDV was inserted between G and L genes of RABV HEP-dG genome and the full-length cDNA plasmid pHEP-dG(H) was constructed.The plasmid pHEP-dG(H) and help plasmid were co-transfected into BHK cell and the recombinant RABV carrying double G and H genes was rescued.The recombinant virus was inoculated into BHK cells to analyze the diffusion ability of the virus.Mice were inoculated with the recombinant virus, and the pathogenicity and immunogenicity of the virus were analyzed.【Result】 RT-PCR and direct immunofluorescence showed that H gene could still be detected in the third generation virus, indicating that the CDV H gene had been successfully inserted into the RABV genome and stably inherited and correctly expressed in HEP-dG(H).The growth curves of HEP-dG(H) virus and HEP-dG strain maintained similar and the titers of two viruses both reached the peak at 96 h.But the titer of recombinant virus HEP-dG(H) was slightly lower than that of HEP-dG at each time point.Spread and infection experiments showed when the multiplicity of infection (MOI) was 0.005, proliferation ability of HEP-dG(H) was lower than HEP-dG strain.HEP-dG(H) and HEP-dG were not lethal to 6 weeks adult mice, however, HEP-dG(H) had a weaker effect on the body weight of adult mice than HEP-dG.HEP-dG (H) and HEP-dG were both able to induce mice to produce antibody, On the 7th day post infection, antibody reached the level of protection (0.5 EU/mL), and the recombinant virus HEP-dG(H) could also induce neutralizing antibody against CDV.Virus challenge test indicated that antibody induced by HEP-dG(H) and HEP-dG could resist the attack of RABV CVS-24 after immunization for three weeks.【Conclusion】 In this study, the recombinant RABV HEP-dG(H) was successfully constructed, which had good immunogenicity and safety, and could be used as a new dual genetic engineering candidate vaccine of RABV-CDV.

Key words: Rabies virus (RABV); Canine distemper virus (CDV); H gene; rabies inactivated vaccine(dG); immunogenicity

中图分类号:

S852.65^＋5

赵静, 褚颖, 罗均, 郭霄峰. 嵌合犬瘟热病毒H基因的重组狂犬病病毒的构建及免疫原性研究[J]. 中国畜牧兽医, 2022, 49(12): 4776-4785.

ZHAO Jing, CHU Ying, LUO Jun, GUO Xiaofeng. Construction and Immunogenicity of Recombinant Rabies Virus with Canine Distemper Virus H Gene[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4776-4785.

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